The invention relates to a method for producing human cell-lines; and cells and cell-lines when produced by such a method.
It is widely acknowledged that it would be advantageous to have in vitro cell models that simulate in vivo conditions. Ideally, the cell models should be able to propagate in culture, express specialised tissue functions and allow fundamental biological problems to be answered by a simple manipulation of the culture conditions. It is therefore not surprising to discover that researchers have spent many years trying to perfect in vitro cell models and in doing so they have discovered that normal differentiated cells generally do not proliferate in culture and often cease to express their specialised function. Indeed as far ago as 1965 Leonard Hayflick reported that when human lung fibroblast are observed in tissue culture the number of divisions these cells can undergo is limited. Similar observations have been made for a wide variety of tissue types and indeed it has been discovered that each type of tissue or cell undergoes a characteristic number of divisions before cell senescence or apoptosis. In order to circumvent what would seem to be age-related cellular death or senescence, researchers have investigated aberrant tumour cell-lines that are capable of growth in culture well beyond the normal level of growth encountered for a normal cell of the same tissue type, that is to say the cells are immortalised. Advantageously, these imortalised cells may retain the ability to express tissue-specific functions. It would therefore seem that immortalised cells may be favourable tools for in vitro investigations.
Indeed, historically the generation of cell-lines was founded on the observation that tumour cells do not exhibit apoptosis. Thus early cell-lines were obtained only as tumour cells or spontaneously immortalised variants of cells which grew readily in tissue culture. Subsequently, the discovery that certain viral oncogenes had the capacity to confer indefinite growth upon various normal cell types led to the rapid generation of non-human cell-lines by transfection of these immortalising genes directly into desired normal cell types in vitro. Immortalising genes can be introduced into cells by a variety of strategies such as transfection and retroviral mediated gene insertions. Thus the use of immortalising genes has facilitated the provision of a wide variety of non-human cell-lines from different tissues.
Over the past fifteen years it has been possible to produce non-human cell-lines retaining differentiated functions by transforming normal cells with chemical carcinogens (1), oncogenes (3), and tumour viruses (4,5). Workers have also attempted to produce human cell-lines retaining differentiated functions using oncogenes (2) and tumour viruses (6). However, although it is possible to produce human cell-lines that have retained some differentiated functions these human cell-lines do not go beyond a few replications before apoptosis or senescence. It therefore follows that such cell-lines are of little value for in vitro investigations.
In view of the considerable success experienced in producing non-human cell-lines it is both puzzling and frustrating that, so far, it has not been possible to use the same techniques to produce successfully human cell-lines, by the term successfully we mean immortalised cell-lines which retain their tissue specific characteristics. It will be apparent, that in the absence of immortalization and tissue specific characteristics cell-lines generated cannot be used as reliable in vitro cell models.
It is interesting to note that the production of immortalised murine cell-lines can be provided using any of the above techniques, whereas it is not possible to provide immortalised human cell-lies. The difference may, in part, be related to the life expectancy of the organism from which the cells derive. For example, the life expectancy of a mouse is approximately 2 years whereas the life expectancy of a human is approximately 70-80 years and therefore it is possible that because of this significant difference in life expectancy there may be more stringent regulation of human cell replication and this stringent regulation may, in part, be responsible for the profound general lack of success in producing differentiated human cell-lines.
Our invention is based on a surprising discovery, we have found that, contrary to expectations, it is possible to produce an immortalised human cell-line which expresses tissue specific functions when the method of the invention is practised, which method involves the use of immature, undifferentiated or precursor cells. Although such cells have been used before to study differentiationxe2x80x94no one has before realised that such cells can be used routinely to provide immortalised human cell-lines that express the tissue specific functions seen in the mature differentiated phenotype.
It is therefore important to note that although undifferentiated cells have been used to provide cell-lines for the purpose of studying the differentiation process where one would expect to start with an undifferentiated cell if one wanted to study the process leading to differentiation, no-one has thought to use undifferentiated cells as a source for providing a cell-line when one simply wants to study the differentiated cell. Rather, it is customary to take a differentiated cell and then imortalised the differentiated cell with a view to producing a human cell-line. It is therefore interesting to note that the method of the invention goes against conventional teaching.
It is also interesting to note that when undifferentiated cells are used to produce human cell-lines for the purpose of studying the process of differentiation and when a controllable immortalising agent has been used such as the SV40 large-tumour T antigen the method has always involved the switching on and off of the immortalising agent at preselected intervals along the differentiation pathway so that at these predetermined intervals the products of differentiation can be identified with a view to establishing markers for mapping the differentiation pathway. In contrast, the method of the invention concerns the use of an undifferentiated cell which is allowed to progress continuously towards terminal differentiation with a view to investigating the differentiated cell therefore, once again, it can be seen that the method of the invention goes against conventional teaching.
It can therefore be seen that there is a need to provide immortalised human cell-lines which can be used as in vitro cell models and it is therefore an object of the invention to provide a method that produces such cell-lines; and cells and cell-lines when produced by such method.
According to a first aspect of the invention there is therefore provided a method for producing human cell-lines, the method comprising;
a) immortalising a human undifferentiated or precursor cell of a given tissue type using an immortalising agent which includes or has associated therewith a control means whereby activation of the control means terminates immortalisation and allows differentiation of the undifferentiated or precursor cell,
b) culturing said immortalised cell in order to produce a homogeneous population of human cells,
c) activating the control means in order to terminate immortalisation and activate differentiation, and
d) allowing differentiation of said cells so as to produce fully differentiated cells of said given tissue type.
It can be seen from the above that the method is characterised by the use of undifferentiated or precursor cells in order to produce a desired fully differentiated human cell-line. It follows that the choice of the undifferentiated or precursor cell will determine the nature of the cell-line. Thus for example, an osteoblast cell-line will be provided by the use of bone marrow stromal cells; an osteoclast cell-line will be provided by the use of haemapoietically derived osteoclast precursors, a heart cell-line will be produced by the use of myocardial precursor cells; a kidney cell-line will be provided by the use of kidney cell precursor cells; a muscle cell-line will be provided by the use of muscle precursor cells; a skin cell-line will be provided by the use of epithelial precursor cells; a liver cell-line will be provided by hepatocyte precursor cells; a lung cell-line will be provided by lung cell precursor cells; and T and B Lymphocytes will be provided be the use of lymphocyte stem cells. It can therefore be seen from the afore examples that the nature of a given cell-line can be determined having regard to the type of differentiated or precursor cells used in the method of the invention.
We have surprisingly found that the use of undifferentiated or precursor cells in the method of the invention provides for an immortalised human cell-line that retains the functional characteristics associated with the cell type from which the cell-line was derived. We have therefore, uniquely, been able to provide human cell-lines for use as in vitro cell models. Our cell-lines are immortal and reliable.
In a preferred embodiment of the invention immortalisation is achieved by using conventional transfection techniques and preferably the immortalising agent is an immortalising gene that is an oncogene, more preferably still, the immortalising agent is a viral oncogene which can be stably integrated into the host cell genome.
Ideally, the immortalisation agent is a construct, preferably a retroviral construct, including an oncogene which oncogene may be virally derived or a human derived oncogene. Any known oncogene may be used such as myc, ras, src, etc.
Alternatively, immortalisation may be effected using physical or chemical means For example, immortalisation may be effected by exposing said cell to radiation or chemicals which are known to promote cell division well beyond the normal level encountered when a cell is not exposed to said physical or chemical means.
In a preferred embodiment of the invention the control means is responsive to environmental conditions such as temperature, pH or ionic concentrations.
In yet a further preferred embodiment of the invention the immortalising agent and control means are integrated, that is to say the immortalising agent is itself controllable. Thus the immortalisation agent and the control means may comprise, for example, a single entity such as a temperature sensitive oncogene. Alternatively, the immortalisation agent and the control means may be two independent entities but in either case, ideally activation/deactivation of the control means has ideally a direct effect such, as in one embodiment, a reciprocal effect on the immortalisation agent. For example, when the control means is activated the immortalisation agent is deactivated. Conversely, when the control means is deactivated the immortalisation agent is activated. Ideally control can be achieved having regard to culture or environmental conditions, for example, in the preferred embodiment of the invention, the immortalising agent is temperature sensitive and the control is thus represented by a temperature sensitive switch so that at about, above or below a first given temperature the immortalising agent is activated so as to immortalise the selected cell type, but at, about, or above a second temperature the immortalising agent is deactivated and in this instance immortalisation terminates and differentiation is allowed to proceed in order to provide a homogeneous population of cells of a given cell type.
Preferably the immortalising agent is the SV40 T antigen which is permissive, that is to say the viral gene is expressed in an active form, at 33xc2x0 C. and non permissive, that is to say the viral gene is expressed in an inactive form at 39xc2x0 C. thus cells immortalised using this agent are temperature sensitive for differentiation.
Uniquely, our cells, when transformed using SV40 T antigen differentiated at the non permissive temperature and survived crisis a condition which is typically followed by apoptosis. In surviving crisis our cells were immortalised. We believe that the feature of immortalisation is due to the use of the undifferentiated or precursor cell in the method of the invention.
In a preferred embodiment allowing differentiation of said cells comprises culturing said cells in the presence of a differentiating agent for example to produce osteoblasts, said differentiating agent is Vitamin D3, ideally in the presence of Vitamin K.
In an alternative preferred embodiment allowing differentiation of said cells comprises culturing said cells in the presence of a differentiating agent for example to produce osteoblasts said differentiating agent is dexamethasone.
In a preferred embodiment allowing differentiation of said cells comprises culturing said cells in the presence of a differentiating agent for example to produce adipocytes, said differentiating agent is rabbit serum or an extract thereof.
In yet a further preferred embodiment of the invention said human cell-line also includes a safety feature which allows for selective disabling or destruction of said cell-line. This safety feature is of advantage where the cell-line is to be used for the purpose of transplantation or is otherwise, whether it be permanently or temporally, attached to, or administered to, or stored in, an individual. The safety feature allows the cell-line to be selectively disabled, and by this we mean rendered harmless, or destroyed, in instances where the cell-line is thought likely to, or is shown to, have the potential to become tumourigenic in vivo, or is thought to be in anyway harmful to an individual.
Preferably the safety feature comprises a gene whose products acts either directly or in-directly to disarm or destroy the cell-line. For example, the gene may be a gene which in the presence of certain agents such as for example anti-viral agents, produces a cytotoxic product. One example of such a gene would be the gene encoding viral thymidine kinase (vTK). This gene avidly converts prescribed anti-viral drugs into cytotoxic intermediates. Another example of a gene which could be used as a safety feature is the cytosine deaminase (CD) gene. The product of this gene renders cells vulnerable to the effects of 5-fluorocytosine and results in cell death.
In a preferred embodiment of the invention the safety feature is expressed in conjunction with the immortalising oncogene. This arrangement is preferred because it means that the immortalising gene is unlikely to be expressed in the absence of the safety feature and visa versa. Our co-pending patent application GB 94 22236.1 teaches how a vector can be produced which provides for co-expression of the safety feature which could be linked with the immortalising oncogene.
In one preferred embodiment of the invention the safety feature gene is placed downstream of the immortalising oncogene and ideally next to but 3xe2x80x2 to, for example, a poliovirus derived internal ribosomal entry site sequence (IRES). This arrangement ensures that the promoter/enhancer elements(s) controlling the transcription of the immortalising oncogene, equally, control the transcription of the safety feature.
It will be apparent to those skilled in the art that other arrangements may be provided in order to enable co-expression of the immortalising oncogene and the safety feature and it is not intended that the above example should be construed in a fashion which limits the scope of protection provided by the application.
According to a further aspect of the invention there is provided a method for the production of osteoblast cells comprising exposing the Human Cell-lines in accordance with the invention to a differentiating agent.
Preferably said differentiating agent is Vitamin D3, ideally in the presence of Vitamin K.
Preferably said differentiating agent is dexamethasone.
According to a further aspect of the invention there is provided a method for the production of adipocytes from Human Cell-lines in accordance with the invention comprising using a differentiating agent.
Preferably said differentiating agent is rabbit serum or an extract thereof.
According to a yet further aspect of the invention there is provided a method for identifying an agent responsible for stimulating differentiation to produce adipocytes the method comprising exposing said Human Cell-line in accordance with the invention to said agent and observing any characteristics of the differentiated phenotype.
Preferably said agent is produced by extraction from rabbit serum using a separation technique, for example, ionic separation, chromatography, protein precipitation etc.
According to a futher aspect of the invention there is provided the use of rabbit serum to produce adipocytes from either a cell-line, preferably a bone marrow cell-line, or at least one precursor cell, preferably a bone marrow precursor cell.
According to a further aspect of the invention there is provided a composition which contains an agent which affects a differentiation process. Preferably said composition is a pharmaceutical composition.
Preferably said agent stimulates the process of differentiation.
Alternatively said agent blocks or prevents differentiation, which differentiation results in the provision of adipocytes.
The bone marrows of osteoporotic patients often contain fatty tissue which is often referred to as fatty marrows. This observation, along with our recognition that the same human bone marrow stromal cells can derive both adipocytes and osteoblasts allows us to propose that osteoporosis may be a consequence of perturbations in bone marrow stromal cell differentiation to the osteoblast lineage, in favour of adipocyte formation. The information provided here, therefore, gives direction to the man skilled in the art to look at this differentiation process for the provision of pharmaceutical agents capable of controlling it. For example:- separation agents from the separation (ionically, HPLC, alternative chromatography, protein precipitation and separation etc.) of normal rabbit serum can be used to determine the agent or agents inducing differentiation to the adipocyte lineage These agents can be used to recognise the intracellular signl transduction pathway involved in the differentiation process; and the subsequent development of agents to effect this pathway. It is envisaged that this information may also lead to the identification of agents for controlling obesity.
According to a further aspect of the invention there is provided cells or cell-lines produced in accordance with the method of the invention. Accordingly there is provided at least one homogenous population of immortalised human cells provided with means to terminate immortalisation such that a homogenous population of differentiated human cells is provided.
According to a yet further aspect of the invention there is provided use of immature, undifferentiated or precursor cells to produce terminally differentiated human cell-lines that express tissue-specific functions.